Blood coagulation is a process consisting of a complex interaction of various blood components or factors which eventually gives rise to a fibrin clot. Generally, the blood components which participate in what has been referred to as the coagulation “cascade” are proenzymes or zymogens, enzymatically inactive proteins which are converted to proteolytic enzymes by the action of an activator, itself an activated clotting factor. Coagulation factors which have undergone such a conversion are generally referred to as “activated factors,” and are designated by the addition of a lower case postscript “a” (e.g., VIIa).
There are two separate systems which can promote blood clotting and thereby participate in normal haemostasis. These systems have been referred to as the intrinsic and the extrinsic coagulation pathways. The intrinsic pathway refers to those reactions which lead to thrombin formation through utilization of factors present only in plasma. An intermediate event in the intrinsic pathway is the activation of Factor IX to Factor IXa, a reaction catalyzed by Factor XIa and calcium ions. Factor IXa then participates in the activation of Factor X in the presence of Factor VIIIa, phospholipid and calcium ions. The extrinsic pathway involves plasma factors as well as components present in tissue extracts. Factor VII, one of the proenzymes referred to above, participates in the extrinsic pathway of blood coagulation by converting (upon its activation to VIIa) Factor X to Xa in the presence of tissue factor and calcium ions. Factor Xa in turn then converts prothrombin to thrombin in the presence of Factor Va, calcium ions and phospholipid. Because the activation of Factor X to Factor Xa is an event shared by both the intrinsic and extrinsic pathways, Factor VIIa can be used for the treatment of patients with deficiencies or inhibitors of Factor VIII (Thomas, U.S. Pat. No. 4,382,083). There is also some evidence to suggest that Factor VIIa may participate in the intrinsic pathway as well (Zur and Nemerson, J. Biol. Chem. 253: 2203-2209, 1978) by playing a role in the activation of Factor IX.
Experimental analysis has revealed that human Factor VII is a single-chain glycoprotein with a molecular weight of approximately 50,000 daltons. In this form, the factor circulates in the blood as an inactive zymogen. Activation of Factor VII to VIIa may be catalyzed by several different plasma proteases, such as Factor XIIa. Activation of Factor VII results in the formation of two polypeptide chains, a heavy chain (Mr=34,000) and a light chain (Mr=24,000), held together by at least one disulfide bond. Factor VII may also be activated to VIIa in vitro, for example, by the method disclosed by Thomas in U.S. Pat. No. 4,456,591.
Factor IX circulates in the blood as a single-chain precursor of molecular weight 57,000 and is converted to an active serine protease (Factor IXa) upon cleavage by Factor XIa in the presence of Factor VIII. Factor IXa consists of a light chain and a heavy chain of molecular weights 16,000 and 29,000, respectively.
Current treatment practices for patients having coagulation disorders (e.g., deficiencies of Factor VIII and IX) generally involve replacement therapy with cryoprecipitate or other fractions of human plasma containing enriched levels of a particular factor. These preparations have heretofore been obtained from pooled human plasma, although the preparation of cryoprecipitates requires the use of a relatively large amount of human plasma as starting material.
Therapeutic uses of Factor VII exist in the treatment of individuals exhibiting a deficiency in Factor VII, as well as Factor VIII and Factor IX deficient populations, and individuals with Von Willebrand's disease. More specifically, individuals receiving Factors VIII and IX in replacement therapy frequently develop antibodies to these proteins. Continuing treatment is exceedingly difficult because of the presence of these antibodies. Patients experiencing this problem are normally treated with an activated prothrombin complex known to consist of a mixture of active and inactive clotting enzymes, including Factor VIIa. Further, recent studies indicate that small amounts (40-50 micrograms) of injected Factor VIIa are effective in controlling serious on-going bleeding episodes in Factor VIII deficient patients who have high levels of antibody in their blood (Hedner and Kisiel, J. Clin. Invest. 71: 1836-1841, 1983).
Due to the diverse sources of the plasma used in the preparation of cryoprecipitates, it is difficult to test the preparations to ensure that they are free of viral contamination. For instance, essentially all recipients of cryoprecipitate show a positive test for hepatitis. Recent reports have also indicated that some hemophiliacs receiving cryoprecipitate have developed acquired immune deficiency syndrome (AIDS). In addition, the purification of large amounts of these factors is extremely difficult and expensive.
Consequently, there exists a need in the art for a method of producing relatively large quantities of pure preparations of Factors VIIa and Factor IX. The present invention fulfills this need through the use of recombinant DNA technology, successfully eliminating the problem of viral contamination and, at the same time, providing a consistent and homogeneous source of active Factor VIIa to treat Factor VIII and Factor IX deficient patients and individuals with Von Willebrand's disease, as well as providing a source of purified Factor IX for use in replacement therapy.